The rearing of nematodes for use in the control of insects and the like was initially effected by culturing the nematodes in their insect host. That was a slow and expensive method of production. A significant improvement in the culturing technology (resulting in reducing the production cost to about one hundredth of the previous cost) is described in the specifications of U.S. Pat. Nos. 4,178,366 and 4,334,498 to R. A. Bedding, and in the specification of the corresponding Australian patent No. 509,879. Those specifications describe three-dimensional in vitro monoxenic culturing techniques which utilise a large surface area and ample interstitial space of a culturing stack. Briefly, those in vitro techniques involve either cutting animal tissues into suitably sized pieces and stacking them appropriately, or forming a stack of inert materials and coating it with an homogenate. The stack so produced is then sterilised by autoclaving and the crude basic medium is made suitable for nematode nutrition and reproduction by culturing the nematode's special symbiotic bacterium (Xenorhabdus sp.) on the medium surface.
The techniques developed by R. A. Bedding, particularly the techniques described in his paper entitled "Large Scale Production, Storage and Transport of the Insect-parasitic Nematodes Neoaplectana spp. and Heterorhabditis spp", which was published in the Annals of Applied Biology, volume 104, pages 117-120, 1984, are very effective. However, the rapid increase in the demand for commercial quantities of entomopathogenic nematodes has revealed the need for a more suitable technique for producing the large quantities of nematodes required for, for example, the treatment of large areas of crops.
Hitherto, the commercial production of nematodes, using a technique described by Bedding in the Annals of Biology paper referred to above, has involved the following sequential steps.
1. Autoclavable polypropylene tubing is sealed with an impulse sealer to make a bag. Autoclave tape is applied over both sides of all seals to provide a bag of adequate strength. Air inlet and outlet tubes are introduced through the tape-reinforced seams and are cemented into place using a silicone rubber solution. A strip of autoclave tape is located centrally on the bag for use as an inoculation panel. PA0 2. Between 3 and 5 kg of waste polyether polyurethane foam is made into crumbs and coated with an homogenate of chicken offal. The polypropylene bag is filled with this medium (during the filling operation the mouth of the bag must be kept clean). The bag is then sealed and the medium is distributed so that it forms an even thickness in the bag. Air is then evacuated from the bag, to enable sterilisation of the medium in minimal time, and the bag is wrapped in hessian, then placed flat in an autoclave. The hessian prevents local burning of the polypropylene bag material. PA0 3. The bag is autoclaved at 121.degree. C. (or at a slightly higher temperature) for a period of from about 75 minutes to about 90 minutes. PA0 4. After cooling for several hours, the bag is placed in a laminar air flow sterile box, then inoculated with a broth suspension of bacteria. The inoculation is usually effected using a small sterilised funnel which is inserted through a slit in the innoculation panel. After adding the bacterial suspension, the slit is sealed using silicone rubber solution and the bag is left for 30 minutes to allow the silicone rubber solution to set. PA0 5. When the silicone solution has set, the bag is kneaded and turned for several minutes to spread the bacterial suspension throughout the medium. PA0 6. The bag is then placed in a constant temperature room, where air is supplied to the interior of the bag through the inlet and outlet tubes, the air being supplied through bacterial filters. After monitoring the air flow for a few hours to ensure that the bag is not over-inflating, the bag is left with the filtered air passing through it for four to five days, to incubate the bacteria. PA0 7. At the end of the incubation period, the bag is inoculated with a nematode culture. The inoculation is effected by pouring the contents of three or four 500 ml flasks of mature nematode culture medium into the bag under sterile conditions, and distributing the culture medium over the surface of the bag contents without undue mixing. Normally the sterile conditions are ensured by performing the inoculation within a laminar air flow through a sterile box after the bag has been thoroughly sprayed with alcohol. The inoculation panel is slit so that a 5 cm diameter funnel tube can be inserted into the slit. The solid culture medium is shaken via the funnel tube into the bag, care being taken to direct the inoculum as widely as possible over the fresh bag medium surface. After the inoculation, the slit is sealed with silicone rubber solution and extreme care is taken during the next 30 minutes to avoid disturbing the slit until the rubber solution has set. PA0 8. When the rubber solution which is sealing the inoculation slit has set, the bag is again connected to the air supply via bacteriological filters. After again observing the bag to ensure that the correct air supply rate has been established, the bag is left in a constant temperature room for two to three weeks. PA0 9. The nematodes are then harvested from the bag by emptying the contents of the bag into a sieve having a floor of fine stainless steel mesh. The sieve containing the culture medium is placed in a slightly larger tray and the culture medium is covered with water for about two hours. During this time, the nematodes leave the culture and go into suspension in the water. The contents of the sieve are then rejected and the liquid remaining is pumped into a settling tank, where the nematodes are repeatedly sedimented and the washing water is decanted. Each bag that undergoes this treatment yields about 0.5 kg of nematodes, together with some debris, provided no bacteria from outside the bag environment have entered the bag after the autoclaving step. The presence of such foreign bacteria will severely reduce the yield of nematodes, and could cause a negligible yield.
The disadvantages of this technique include
(a) the need for a fresh bag to be constructed for each culture; PA1 (b) the requirement for at least two bacterial filters (which are not inexpensive) for each bag (although they can be reused 3 times); PA1 (c) the considerable time that is required for the steps of (i) filling and sealing the bags (which ought to be conducted with two operators), (ii) the evacuation of air, (iii) the inoculation with both bacteria and nematodes, and (iv) waiting for the silicone rubber seal to dry; PA1 (d) the inoculation dispersal is not efficient; PA1 (e) the bags can be rendered useless by blockages in the filters (which occur ocassionally despite monitoring of the inflation and re-inflation of the bags) or by the failure of the electricity supply to the pumps which pump the air through the filters; PA1 (f) the inflation of the bags results in a considerable reduction of the basal surface area unless the bag is held flat with weights; since the depth of the medium in the centre of each bag should not exceed 10 cm, this means that a reduced quantity of medium can be contained in each bag; PA1 (g) there is an uneconomical use of space due to (i) the fact that the inflated bags take up many times the storage volume of the actual culture medium and (ii) the necessity of ready access to all bags for adjustment of air flows; PA1 (h) the bags require suitable shelving to hold them, so that either a supply of air ducted to all shelf areas or a separate air pump for each bag must be provided; and PA1 (i) air pump maintenance and replacement is a significant expense. PA1 (a) a substantially flat tray having a continuous wall or a plurality of contiguous walls extending substantially orthogonally from the periphery of said tray; PA1 (b) a substantially flat lid having essentially the same shape as said tray but dimensions greater than said tray, so that when said lid is positioned to cover said tray, the periphery of said tray is outside said wall or walls; said lid having a continuous side wall or a plurality of contiguous side walls which extend substantially orthogonally from the periphery of said lid; and PA1 (c) a gasket of medium density polyether polyurethane foam (or a similar foam material) positioned around the edge of said lid within and immediately adjacent to said lid side wall or walls, whereby, when said lid is placed on said tray, said gasket is contacted by the upper edges of the tray wall or walls. PA1 (a) coating crumbed polyether polyurethane foam (or similar material) with an offal homogenate, and distributing the coated, crumbed foam evenly over the base of a tray of a culture vessel constructed in accordance with the present invention, then placing the lid of the culture vessel over the tray so that the foam gasket thereof is in contact with the entire top of the wall or walls of the tray; PA1 (b) autoclaving the culture vessel and its contents at a temperature of about 121.degree. C. for about 1 hour; PA1 (c) allowing the autoclaved vessel to cool; PA1 (d) inoculating the autoclaved foam and homogenate with the primary form of the symbiotic bacterium, Xenorhabdus sp., of the entomopathogenic nematode species to be cultured; PA1 (e) maintaining the culture vessel and its contents at a temperature of about 23.degree. C. for a period sufficient to incubate the symbiotic bacteria; PA1 (f) inoculating the contents of the culture vessel with a monoxenic suspension of nematodes; PA1 (g) maintaining the culture vessel at a temperature of about 23.degree. C. for a period sufficient to enable the nematode species to reproduce and generate infective juveniles (J3) of the nematode species; and PA1 (h) harvesting the juvenile entomopathogenic nematodes from the culture vessel.
Despite these disadvantages, sufficient nematodes for large scale field trials of up to 50 hectares, requiring the yield of at least two bags per hectare, can be produced. However, the production of sufficient nematodes to treat a commercial crop of thousands of hectares is unlikely to be technically feasible using this technique.